Using structural approaches (cryo-electron microscopy and x-ray crystallography), we will investigate the mechanisms used by coxsackievirus B3 (CVB3) to enter host cells by engaging two receptors (decay accelerating factor and coxsackievirus and adenovirus receptor). CVB3 is a human pathogen that causes myocarditis, pancreatitis, and has been implicated in the onset of juvenile diabetes mellitus. Our proposed studies of CVB3 entry are a continuation of a productive collaboration with Dr. Jeffery Bergelson, Division of Infectious Diseases at Children's Hospital at Philadelphia. A combination of traditional and innovative methods will be used to visualize both receptor binding and the resulting conformational changes that lead to entry and uncoating. Of particular interest is a novel entry intermediate that has never been observed (Aim 1). This particle has been isolated with fingerlike density projections extruding from the capsid, and these densities are hypothesized to be peptides used to anchor the particle to the host membrane. We will investigate the capability of this new particle (and other entry intermediates) to bind receptors and infect the host. In addition to conventional structural studies made with purified virus and recombinant receptor proteins in solution (Aim 2), the full-length receptor will be anchored onto a lipid bilayer disc to form virus-receptor complexes for an in situ asymmetric study (Aim 3). This approach simulates the physiological binding event, in which the virus interacts with receptors at a single, focused region on its capsid. Preliminary results with this asymmetric approach suggest the virus behaves differently when interacting with a few membrane-bound receptors compared to many soluble receptors. Specifically our in situ A-particle retains an intact genome (as the A-particle does in an infection) and undergoes different protein rearrangements compared to particles that are treated with soluble receptors or heat. The proposed study is directed at understanding the structural alterations that an enterovirus capsid must undergo to uncoat after successfully finding and attaching to the receptor in situ.